Long-Range Hairpin Slippage Reconfiguration Dynamics in

Jun 26, 2019 - Trinucleotide repeat (TNR) sequences, which are responsible for several neurodegenerative genetic diseases, fold into hairpins that int...
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Cite This: J. Phys. Chem. Lett. 2019, 10, 3985−3990

Long-Range Hairpin Slippage Reconfiguration Dynamics in Trinucleotide Repeat Sequences Cheng-Wei Ni, Yu-Jie Wei, Yang-I Shen, and I-Ren Lee* Department of Chemistry, National Taiwan Normal University, Taipei 11677, Taiwan

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S Supporting Information *

ABSTRACT: Trinucleotide repeat (TNR) sequences, which are responsible for several neurodegenerative genetic diseases, fold into hairpins that interfere with the protein machinery in replication or repair, thus leading to dynamic mutation ―abnormal expansions of the genome. Despite their high thermodynamic stability, these hairpins can undergo configurational rearrangements, which may be crucial for continuous dynamic mutation. Here, we used CTG repeats as a model system to study their structural dynamics at the single-molecule level. A unique dynamic two-state configuration interchange was discovered over a wide range of odd-numbered CTG repeat sequences. Employing repeat-number-dependent kinetic analysis, we proposed a bulge translocation model, which is driven by the local instability and can be extended reasonably to longer (pathologically relevant) hairpins, implying the potential role in error accumulation in repeat expansion.

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found in pathological samples,26 giving a hint that the paritydependent properties of these hairpins may emerge as the repeat number increases. Our previous work has demonstrated the dynamic hairpin reconfiguration between the overhang and blunt-end configurations in a similar system of pentanucleotide TGGAA repeats and proposed a continuous expansion model.27 The local-instability-driven hairpin slippage plays a crucial role in error-prone expansion by the overhang hairpin momentarily slipping into an error-prone blunt-end configuration. To investigate the detailed mechanism of the dynamic hairpin slippage reconfiguration, here, we select the TNR sequences as our model system owing to its higher pathological importance and, also, the smaller unit size that allows us to perform a wide-range repeat-number-dependent study. We probed the structural dynamics of TNR hairpins using singlemolecule fluorescence resonance energy transfer (smFRET) microscopy. A dynamic configurational rearrangement between the blunt-end and overhang configurations was found in oddnumbered (CTG)n sequences with n ≥ 13. Through repeatnumber-dependent interconversion kinetics, we proposed a bulge-sliding model, which is further supported by our Monte Carlo simulations. The sliding motions rely on local instability and can be extrapolated reasonably to longer (pathologically relevant) repeats regardless of their high global stability and, perhaps, correlate with the accumulation of repeat elongation errors. To probe for size-dependent structural polymorphisms, we designed an assay that carries the capability of resolving blunt-

rinucleotide repeats (TNR) are the most common repetitive DNA sequences and usually serve as hotspots for genome instability.1−4 Slippage of repetitive DNA forms hairpins and other structures that interfere with the replication or repair machinery and lead to abnormal expansions of the genome,2−11 resulting in overexpression of translational or transcriptional products with cytotoxicity to neuron cells and ultimately leading to incurable neurodegenerative diseases.12 These unusual structures, originating from base-pair mismatch and repetitive sequence motifs, usually carry unique structural features that can be recognized by small-molecule ligands for potential disease detection or therapeutic applications.13,14 Among these TNR sequences, CTG/CAG is responsible for the most (over 14) types of neurodegenerative disorders, for example, Huntington’s disease, myotonic dystrophy, and spinocerebellar ataxia (SCA).15,16 Single-stranded CTG and CAG repeats fold into thermodynamically stable stem-loop hairpins.17−19 Nascent TNR hairpin formation is crucial to repeat expansion in polymerase-catalyzed in vitro replication.20 In double-stranded (CTG)n·(CAG)n DNA, a double-hairpin loop of a CTG and CAG tandem repeat may be induced by negative supercoiling or stabilization of the nascent hairpin by protein (e.g., MSH2/MSH3) binding and is thus believed to be the hot spot for repeat expansion.21,22 Recently, a large-scale expansion model involving break-induced replication with an intermediate of stable hairpin formation at double-strand break was proposed.23 Parity-dependent (even/odd) polymorphism and thermodynamic properties were found in (CTG)n hairpins.18,19,24,25 Even- and odd-numbered TNR hairpins fold into the bluntend and overhang configurations, respectively, and a preferred repeat expansion on the blunt-end configuration was suggested.18 However, no evidence of parity dependence was © 2019 American Chemical Society

Received: May 28, 2019 Accepted: June 26, 2019 Published: June 26, 2019 3985

DOI: 10.1021/acs.jpclett.9b01524 J. Phys. Chem. Lett. 2019, 10, 3985−3990

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The Journal of Physical Chemistry Letters end and overhang structures in our smFRET experiments, illustrated in Figure 1a. We tested a wide range of (CTG)n (n =

Figure 1. Single-molecule FRET experiments on tandem CTG and CAG repeats. (a) Single-molecule assay. (b−d) EFRET histograms of (b) (CTG)n and calibration assays (Calib and (CTG)6T3 indicated in Table S1), (c) (CAG)n, and (d) odd-numbered (CTG)n repeats.

Figure 2. Time-dependent single-molecule FRET experiments. (a) Raw Cy3 (green) and Cy5 (red) intensities (top) and their corresponding EFRET as functions of time (bottom), which are overlaid with the HMM fitting result (red curves). (b) Transition density plots extracted from the HMM fits. (c) Separation of the two states using HMM fits. Both histograms are overlaid by the calibration assays (blue: (CTG)6 and red: (CTG)6T3 indicated in Table S1). (d) Reciprocal of rate constants and equilibrium constants against the repeat number. Averaged value and error bars were obtained from three independent experiments. More representative traces are shown in Figure S2. I, intensity; A.U., arbitrary unit.

5−37) and (CAG)n (n = 5−21), and representative results are shown in Figure 1b,c, respectively, while more are depicted in Figure S1. In both cases, we observed a clear parity-dependent alternation in EFRET. An EFRET distribution centered at ∼0.84 for (CTG)4 and other even-numbered (CTG)n were consistent with the expected EFRET of ∼0.84 for the blunt-end configuration, verified by using a mimic calibration assay (inset of Figure 1b). Similar behavior was also observed in (CAG)n (Figure 1c). With the longer odd-numbered (CTG)n, although the alternating behavior still hold true, the EFRET distribution is broadened as the repeat number increases (Figure 1d). This broadening could originate from conformational heterogeneity and possibly from conformational changes within the time span of single-snapshot acquisition (10 frames, ∼300 ms). To clarify, we recorded time-dependent EFRET traces at a rate of 33 s−1. We found that with odd-numbered n ≥ 13, rapid stochastic transitions of EFRET states were observed in the vast majority of the traces, as shown in Figure 2a, while the traces with evennumbered (CTG)n remained virtually steady (Figure 2a top). We employed the Hidden Markov Model (HMM) with an initial guess of 10 possible states to identify the transition between states.28 A clear two-state transition can be seen in the resulting transition density plot (TDP) illustrated in Figure 2b. The vast majority of transitions happen between states of EFRET ∼0.84 and 0.72. These two states were identified as the bluntend and the overhang configurations using their mimic assays, respectively (Figure 2c). With the two-state reversible reaction model, kinetic constants of the forward and reverse reactions were also

obtained from the transition probabilities in the HMM fitting. The mean dwell time (reciprocal of the rate constant) with the corresponding equilibrium constants are plotted against the repeat number, n, in Figure 2d. We found that the mean dwell time increased monotonically within the range of n tested in both the forward and backward directions. Thus, the equilibrium constants remain unchanged over the range we tested. The interconversion of hairpin configurations possibly goes through the complete melting of the hairpin into a random coil state followed by refolding into its conformational counterpart. We can rule out this possibility in our repeat-numberdependent experiment because the melting of the hairpins should be dramatically retarded and becomes infeasible as the activation energy increase nearly monotonically with the repeat number. Therefore, we propose a local instability-driven mechanism: the hairpin first partially opens and forms a hotspot, presumably a bulge, and this hotspot slides, ultimately translocating to the termini where it completes the configuration transformation. To examine this local bulge-sliding model, we designed a series of assays containing altered repeat sequence with discrete CTG·CAG pairings serving as roadblocks in the 3986

DOI: 10.1021/acs.jpclett.9b01524 J. Phys. Chem. Lett. 2019, 10, 3985−3990

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The result of the Hidden Markov analysis clearly shows that the mean dwell time, in both the forward and reverse reactions, rises as the number of roadblocks increases. The nearly monotonic retardations with the number of roadblocks support the bulge-sliding model (Figure 3c). Interestingly, when we placed a single-point T-to-A alteration immediately next to the loop in (CTG)25 (mLoop, Figure 3d), we found that the interconversion was severely retarded. We propose a bulge-sliding model for the interconversion of both the forward (overhang to blunt-end) and reverse (bluntend to overhang) reactions, illustrated in Figure 4. Although the bulge may form anywhere in the stem by thermalfluctuation-driven local melting, the chances of bulge formation near the loop are much higher evident in the severe retardation of structural interconversion in the assay with a single-point alteration near the loop (Figure 3d). Flexibility near the loop facilitates the temporary opening of C·G base pairs, and T·T mismatches adjacent to the 4-nt (overhang) and 3-nt (blunt-end) loops lead to a large loop as the precursor of loop reconfiguration with estimated activation energies of 0.6 and 1.2 kcal mol−1, respectively.29−31 With transient melting of the adjacent unit segment, one possibility is that the CTG· CTG or CTGC·GCTG of antiparallel interactions leads to the formation of a bulge (side loop) and completes the loop reconfiguration. The bulge then translocates through the opening of the adjacent short pairing segment and reforms a new one by translocation of the bulge; this process is highly stem-length (repeat-number) dependent. The activation energies of these processes are expected to be relatively low (1−4kBT) and, hence, accessible solely by thermal fluctuations.29−31 This model is further supported by the excellent agreement of the experimental dwell-time distributions and our results of Monte Carlo simulation using the kinetic scheme of this model (Figure 4c; see the Supporting Information for details). While even-numbered (CTG)n undoubtedly folds into a blunt-end hairpin configuration with a stem and a 4-nt TGCT loop, controversy exists over the configuration of the oddnumbered (CTG)n sequence. The (CTG)5 sequence was assigned to a blunt-end hairpin with a 3-nt CTG loop by the Gupta group using NMR spectroscopy.17 In contrast, (CTG)11

(CTG)25 hairpin (Figure 3a). These roadblocks increase local, as well as global, stability by replacing the T·T mismatches

Figure 3. Time-dependent interconversion observations on the assays containing altered repeat sequence. (a) Illustration of the assays. The CAG alternations are indicated as red lines. (b) Raw Cy3 (green) and Cy5 (red) intensities (top) and their corresponding EFRET as functions of time (bottom), which are overlaid with the HMM fitting result (red curves). (c) Reciprocal of rate constants. Average values and error bars were obtained from three independent experiments. (d) Result from the assay with single-point T-to-A alteration immediately next to the loop. More representative traces are shown in Figure S3.

with T-A Watson−Crick pairings. In a local melting model, multiple roadblocks will serve as multiple rate-determining steps that retard the sliding of the bulge nearly monotonically. With the insertion of up to four discrete roadblocks, the conformational transition between two hairpin configurations can be observed with a retarded rate (Figure 3b), as expected.

Figure 4. Purposed bulge-sliding model for hairpin configuration interconversion. (a) Schematic illustration of the model. (b) Repeat-numberdependent dwell times. Experimental data (reciprocal of the rate constants, squares) are overlaid with optimized Monte Carlo simulation results (lines). (c) Representative dwell time distributions of experimental (bars) and Monte Carlo simulated (lines) results; others are shown in Figure S4. 3987

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However, the slippage motion was neither observed in the tandem CAG repeats nor was it observed in the CCG/CGG repeats. Perhaps the expansion of CAG repeats is coordinated with that of CTG repeats. On the other hand, both even- and odd-numbered CCG/CGG repeat hairpins are in blunt-end configuration (Figure S6). Nevertheless, the replication and repair processes associated with the abnormal expansion of TNR are very dynamic, and recruiting the newly synthesized DNA strand into the hairpin may be crucial in error-prone expansion. These TNR hairpins all carry parity-dependent polymorphisms and local instabilities due to mismatched base pairings. We anticipate that the slippage reconfiguration of the hairpin may allow recruiting of the newly synthesized DNA strand into an enlarged hairpin through reconfiguration of the hairpin and ultimately cause multiple expansions in a single cycle of replication and repair. Moreover, the slippage reconfiguration was only slightly retarded when altered assays were used (Figure 3a,b). This indicates that the slippage reconfiguration is capable of overcoming short segments of perfect Watson−Crick pairs, for example, CTG·CAG, detaching the newly synthesized CTG strand from the template strand and melting into the hairpin. In conclusion, we utilized the (CTG)n TNR repeats as a model system to study the dynamic slippage reconfiguration of the TNR hairpins. We proposed a local-instability-driven bulge-sliding model, where a bulge is initially formed near the loop region and translocates through the stem to accomplish the conformational conversion; this model was further supported by our Monte Carlo simulations. With this hairpin slippage reconfiguration mechanism, the dynamic configuration interconversion can be extended reasonably to longer (pathologically relevant) hairpins. We suggest that slippage reconfiguration dynamics may play a crucial role in an efficient expansion of many TNR sequences.

was identified as a hairpin with a 4-nt TGCT loop and a 3-nt CTG overhang by the Delaney group utilizing the chemical footprinting assay and further supported by their thermodynamic profiles.18,19 Our single-molecule method was able to show the coexistence of both exchangeable configurations. The more favorable blunt-end configuration could dominate in the bulk NMR structural determination, while the temporarily slipped-out overhang can be chemically modified and probed in the chemical footprint assay. In our observations, an equilibrium constant (blunt-end/overhang) was obtained based on the forward and reverse kinetic rates. Given that there are two energetically close and experimentally indistinguishable states in the overhang configuration (Figure S5), a 3′-overhang and a 5′-overhang, we could obtain a Gibbs free energy difference (ΔG) of −0.70 (±0.01) kcal mol−1 of the blunt-end and the overhang configurations. This rather small ΔG allows the coexistence of these two configurations thermodynamically. The overhang configuration of a (CTG)n hairpin consists of a tetranucleotide TGCT loop and a stem formed by a C·G pair and (n − 3)/2 antiparallel CTG·CTG pairs, while the bluntend structure has a stem formed by (n − 1)/2 antiparallel CTG·CTG pairs and a trinucleotide CTG loop. The net difference in stabilizing interactions, a C·G base pairing and a T·T mismatch, give rise to an estimated 1−3 kcal mol−1 lower free energy in the blunt-end configuration.29−31 However, the energy penalty of forming a relatively unstable trinucleotide loop lowers the value of ΔG and makes the blunt-end configuration only slightly favorable. In comparison, replacement with A·A mismatches in the odd-numbered (CAG)n repeats increases the free energy of the blunt-end configuration by ∼0.5 kcal mol−1,29−31 leading to an expected population ratio of 1:0.7 in slight favor of the overhang configuration. However, we did not capture any blunt-end configuration state (EFRET ∼ 0.84) in the (CAG)n repeats we probed. Possibly, the CAG·CAG antiparallel interaction is too weak to hold an unstable 3-nt CAG loop stably,32 resulting in a much energetically unfavored 7-nt dAGCAGCA loop. In the evennumber cases, the blunt-end configuration consists of a longer stem (with an additional C·G pair) and a more stable tetranucleotide TGCT (or AGCA) loop compared with the overhang counterpart. The large difference in free energy restricts the blunt-end configuration to even-numbered repeats of (CTG)n (or (CAG)n). Previously, we reported on parity-dependent polymorphism and slippage dynamics in the hairpins of TGGAA repeats associated with SCA-31 diseases and proposed a continuous repeat expansion model.27 The slippage hairpin reconfiguration from the overhang to the blunt-end configuration, which has the potential to induce repeat expansion in replication, recombination, or repair, is crucial to continuous expansion. Through a repeat-length-dependent study of CTG repeats, we suggested that the long-range slippage hairpin reconfiguration can be achieved through local-instability-driven bulge translocations; thus, regardless of the increasing global stability, the slippage motion can be reasonably extrapolated into even longer repeats in the premutation and pathological ranges. In reality, the forming of multiple bulges simultaneously (branched hairpin) in a longer repeat sequence can be expected and complicates the kinetics scheme. In these cases, we expect the reconfiguration between the overhang and bluntend structures can be accelerated due to the shorter bulgesliding distance required to accomplishing the reconfiguration.

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EXPERIMENTAL AND COMPUTATIONAL METHODS Partial duplex DNA samples used in the smFRET experiments were prepared by annealing two oligonucleotides at concentrations of ∼5 μM in the polymerase chain reaction (PCR) thermal cycler with an initial heating step at 95 °C for 5 min followed by gradual cooling to room temperature (22 °C) at a rate of −1 °C/min. The oligonucleotides used are listed in Table S1. Before annealing, each oligonucleotide of interest (those with an NH2C6 modification) was first reacted overnight with sulfo-Cyanine3(Cy3)-NHS ester (Lumiprobe) at room temperature, followed by two runs of ethanol precipitation to remove the unreacted sulfo-Cy3-NHS ester. Prior to use, the annealed partial duplex DNA was diluted to a concentration of ∼10 pM with wash buffer (20 mM Tris, 100 mM NaCl, pH 7.8), and the diluted solutions were heated to 50 °C followed by slow cooling to 22 °C at a rate of −1 °C/12 min in a PCR thermal cycler to ensure that they reached thermodynamic equilibrium and to suppress interstrand dimer formation. The partial duplex DNA of interest (∼10 pM in wash buffer) was then introduced to the fludic chamber for 2 min to allow tethering of the DNA onto the surface-anchored Neutravidin and then flushed with wash buffer to remove the excess sample. The fluidic chamber was built with a quartz microscope slide, double-sided tape, and a glass coverslip in a sandwich configuration. The inner surface of the chamber was pretreated and functionalized with a mixture of polyethylene glycol (PEG, 3988

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tion of graphical processing unit (GPU)-accelerated parallel computing. A random number was generated for each time stamp (1 μs) and used to determine the direction of the elementary reaction step (or no reaction). We simulated 10 000 individual molecules for each repeat number n and recorded their dwell time until the conformation change was completed. We then calculated the mean dwell time of these molecules. The sum of squared deviations of dwell time between the simulated and experimental data across n was then minimized. See the Supporting Information for more details.

MW = 5000, LaysanBio) and biotinylated PEG (MW = 5000, LaysanBio) and stored in a light-proof vacuum tube at −20 °C. Immediately before the experiment, Neutravidin (0.2 nM in wash buffer, Thermo) was introduced to the chamber for 1 min to allow Neutravidin to bind to biotin on the functionalized surface and then flushed with wash buffer. The sample assays were then placed onto a home-built smFRET apparatus described elsewhere.27,33 Briefly, surfacetethered single molecules in the chamber were excited by the evanescent field produced by the total internal reflection at the interface of the quartz slide and the solution using 532 nm (LASOS DPSSL-533, Germany) or 633 nm (Omicron Laser LuxX633, Germany) continuous-wave lasers. The resulting fluorescence images were collected by the objective (Nikon CFI Plan Apo VC 60XWI, Japan) on a commercial microscope (Nikon Eclipse Ti:S, Japan) with optical filters (Semrock LPD01-532RS-25, BLP01-532R-25, and NF03-633E-25) to remove scattering from the excitation lasers and finally directed to a home-built dual-view optical setup, where the image was cropped, enlarged (1.5×) and relayed to project onto an electron-magnified charge-coupled device (EMCCD, Andor iXon Ultra 897, U.K.) detector. A dichroic mirror (Semrock FF640-FDi01) was placed in the setup to separate and offset the Cy3 and Cy5 fluorescence and arrange the projected images in a side-by-side configuration on the detector to allow simultaneous detection of the Cy3 and Cy5 channels. Prior to the experiment, the wash buffer in the sample chamber was replaced by the image buffer containing a pyranose oxidase and catalase (POC)-based oxygen scavenger system to avoid blinking and prolong the life span of the dye molecules before photobleaching occurred.34 The recipe for the image buffer is given in Table S2. We used the alternating-laser-excitation (ALEX) technique to obtain EFRET distribution snapshots.35 A 30-frame movie clip (at ∼33 frames per second (fps)) was recorded for each field of view using 532 nm laser excitation for the first 20 frames and the 633 nm laser for the remaining frames. An accumulated image was built by summing the third through the 12th frames for each clip and used for single-molecule identification,33 and the EFRET for each molecule was calculated according to the following equation: EFRET = (IA − αID)/(IA + ID), where ID and IA are the intensities from the donor and acceptor channel and α is the donor leakage correction factor (0.2 in this experiment). The accumulated intensity from the acceptor channel between the 23rd through the 28th frames (633 nm excitation) was used to validate the status of the Cy5 fluorophore. A threshold was set to eject those molecules with missing or photobleached Cy5 fluorophores. For each sample, we repeat this experiment 20−30 times at different fields of view to accumulate a sufficient number of molecules for analysis. We also recorded movie clips of 2000 frames (33 fps) with 532 nm excitation for dynamic studies. Single-molecule identification was performed in the same manner as described in the previous paragraph. Time-dependent EFRET traces were calculated from the intensities of the donor and acceptor channels. The obtained traces were manually screened and trimmed to remove those affected by unwanted photophysical activities (e.g., photobleaching and blinking). These traces were then used for HMM fittings using the HaMMy program.28 Kinetic Monte Carlo simulations were carried using our home-built Matlab (Mathworks) script with the implementa-

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.9b01524.

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Details of the kinetic Monte Carlo simulations; oligonucleotide list; POC oxygen scavenger system; parameters for Monte Carlo simulations; additional experimental and simulation results. (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: +886-2-7734-6115. Fax: +886-2-2932-4249. E-mail: [email protected]. ORCID

I-Ren Lee: 0000-0001-5655-6049 Notes

The authors declare no competing financial interest. Kinetic Monte Carlo simulation scripts written in Matlab (Mathworks) with the implementation of the graphical processing unit (GPU) accelerated parallel computing can be found on Github (https://github.com/SingleMoleculeTW/ gKMC).

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ACKNOWLEDGMENTS We thank Prof. Ming-Hon Hou and Dr. Chung-ke Chang for helpful discussions. This work was supported by Grant 1052113-M-003-009-MY2 from the Ministry of Science and Technology, Taiwan.

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REFERENCES

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DOI: 10.1021/acs.jpclett.9b01524 J. Phys. Chem. Lett. 2019, 10, 3985−3990